The present invention relates to a method for producing gradient gel medium membrane for electrophoresis for determining the base sequence of DNA or DNA partially decomposed material.
Conventionally, in a plane electrophoretic method, high-molecular concentration radient el of acrylamide having no self-supporting property has been obtained by forming gel in layers having concentrations different from each other and stacked on one support or between two supports in the electrophoretic direction. Such a gel has been used as a membranous material.
In the method in which gel is formed on a support, however, there has been a disadvantage in that the gel can sometimes be damaged by dropping a material other than a sample onto the gel while the gel is being formed on the support, when the gel is set in an electrophoretic tank, when the gel is in a state of preservation, or when the gel is being added with a sample to be analyzed. Therefore, close attention and a high level of skill are required in operation.
On the other hand, in a vertical electrophoretic method in which gel is formed in a mold formed by two glass plates or the like, and electrophoretic analysis is performed while holding the mold vertically, there has been a disadvantage in that it is difficult to make the mold uniform in thickness and a high level of skill is required in operation to pour a gel forming solution into a narrow mold before the gel forming solution has gelled.
Particularly, in an operation for determining the base sequence of DNA, it is desirable to form an elongated sheet of gel so that pieces of DNA, as many as possible in number, can be analyzed using one sheet of gel. However, production and handling of such an elongated sheet of gel has proved difficult. Further, since glass plates are used, there has been a disadvantage in that the glass plates are apt to break.
Recently, for the purpose of industrially producing a gradient gel material for electrophoresis, there has been disclosed a method for producing gradient gel including steps of: preparing an aqueous solution or a water dispersion liquid of a mixture of acrylic amide monomers and a cross-linking agent, adding a free radical generating material for initiating polymerization of the monomers by absorption of light irradiated on the solution, forming the solution into the shape of desired gel product, and adjusting the time period of light irradiation on the monomer solution so as to change the porosity of the gel while the solution is being irradiated so as to cause polymerization and cross-linking in the monomer solution or the monomer dispersing liquid. (See European Patent No. 0169397A).
Further, there has been disclosed a method for producing gradient gel for electrophoresis having a concentration gradient of a polymer in the electrophoresis direction and including steps of: supplying two kinds of aqueous solutions different in concentration from each other and each containing monomers, a cross-linking agent and a polymerization initiator into a forming device while mixing the aqueous solutions with a mixing ratio therebetween being changed gradually, and completing polymerization of the monomers with the cross-linking agent in the forming device. (See Japanese Patent Publications Nos. 61-022903 and 61-39617).
The former method, however has problems in that the cost is high due to the need for light irradiation equipment and generally low productivity, the resolution is low because of an increased thickness of the gel, and it is impossible to obtain gradient el having good stability and reproducibility because the reaction initiator causes polymerizing and cross-linking reactions in the presence of light even after completion of prosecution. The latter method, on the other hand, has problems in that the productivity is poor because of its batch type production system, and it is difficult to uniformly branch a medium liquid for electrophoretic separation when it is poured into forming devices, making it impossible to obtain gradient gel with good reproducibility.
To produce gradient gel medium membrane for electrophoresis with a high productivity and good reproducibility and to solve the foregoing problems, the present applicant has proposed a method for producing gradient gel medium membrane for electrophoresis including steps of: mixing high and low concentration monomer solutions supplied at respective flow rates, the ratio of which is continuously changed, with a predetermined quantity of polymerizing reaction initiator solution with a static mixer to thereby prepare a gel forming solution for use in electrophoresis, and coating a continuously moving web with the gel forming solution for electrophoresis.
In this proposed method, however, the flow-rate ratio of the high and low concentration monomer solutions is changed so that the concentration gradient changes from low to high every product unit length as shown in FIG. 3A, and therefore the distance t at the changeover length for every product unit length has been exceedingly long, that is, t=1.0 m for l=0.4 m.